Caracterización de la interacción entre proteínas CKAMP y receptores de glutamato tipo AMPA

Los receptores AMPA (AMPAR) son canales iónicos que median la mayor parte de la neurotransmisión excitatoria rápida en el sistema nervioso central y participan en la plasticidad neuronal. Los AMPAR contienen un núcleo tetramérico compuesto por diferentes combinaciones de las subunidades GluA y más de 30 subunidades auxiliares diferentes que generan un amplio grupo de complejos. La composición de los complejos determina las propiedades del receptor, incluyendo la expresión en la sinapsis, el tráfico celular, la farmacología o a las propiedades cinéticas. Las CKAMP son una familia de 4 proteínas (CKAMP39, CKAMP44, CKAMP52 y CKAMP59) recientemente identificadas como subunidades auxiliares del AMPAR. Hasta el momento no se ha realizado ningún estudio estructural de la interacción AMPAR-CKAMP, cuya topología transmembrana difiere del resto de subunidades auxiliares, ni se ha explorado la composición y estequiometria de los complejos que las contienen. En este proyecto se presenta el clonaje de las CKAMP mediante el método «IVA cloning», los ensayos de expresión y purificación de CKAMP, así como los intentos de aislar los complejos AMPAR-CKAMP para su posterior estudio estructural.<br /

Caracterización bioquímica y estructural de complejos de receptores AMPA y subunidades auxiliares TARP tipo II

Los receptores de glutamato tipo AMPA (AMPAR) son canales iónicos localizados en la sinapsis, donde juegan un papel fundamental en la neurotransmisión excitatoria y plasticidad neuronal. Los AMPAR son complejos tetraméricos ensamblados a partir de cuatro subunidades (GluA1-4), codificadas por diferentes genes (GRIA-4), en forma de dímero de dímeros. Los receptores AMPAR son impermeables a Ca2+ cuando contienen la subunidad GluA2, pero permeables a Ca2+ cuando contienen combinaciones del resto de subunidades (GluA1, GluA3 y GluA4). Estos últimos pese a ser menos abundantes en el cerebro, tienen un papel importante en diversas situaciones patológicas (infarto cerebral o esclerosis lateralamiotrófica). Por otro lado, existen más de 30 subunidades auxiliares que interaccionan con los AMPAR, aumentando su variabilidad estructural y funcional. Las TARP constituyen una de las familias más representativas de subunidades auxiliares y se pueden clasificar en tipo Ia, Ib y tipo II. Aunque se dispone de mucha información sobre la función, estructura y regulación de las TARP tipo I, las TARP tipo II han sido poco caracterizadas. En este trabajo se profundizó en la estructura entre los AMPAR permeables a Ca2+ y las TARP tipo II, explorando la estabilización del complejo y su producción a gran escala en células de mamífero. La purificación del complejo permitió iniciar una caracterización estructural preliminar mediante microscopía electrónica de tinción negativa.<br /

Secreción inadecuada de hormona antidiurética secundaria a estesioneuroblastoma

Presentación Se presenta un caso de síndrome de secreción inadecuada de ADH (antidiuretic hormone) de origen paraneoplásico secundario a un estesioneuroblastoma o neuroblastoma olfatorio. La secreción inadecuada de ADH es la causa más frecuente de hiponatremia euvolémica en la práctica clínica. Los criterios diagnósticos son: sodio plasmático menor a 135 mEq/L, osmolaridad plasmática disminuida, volumen extracelular normal, osmolaridad urinaria mayor a 100 Osm/Kg, sodio urinario mayor a 40 mEq/L, ausencia de insuficiencia suprarrenal o hipotiroidismo. Como síndrome paraneoplásico se ha asociado a carcinoma microcítico de pulmón, mesotelioma, timoma, duodenal, pancreático, gástrico, síndromes mieloproliferativos y tumores cerebrales. En nuestro caso se asocia con un tumor nasofarígeo, el neuroblastoma, un tumor originado en el neuroepitelio olfatorio de la placa cribiforme a nivel del tercio superior del septo nasal y puede extenderse hacia el espacio intracraneal. Predomina en el sexo masculino, y tiene una distribución bimodal en la edad de presentación, (segunda y sexta décadas). Presentan crecimiento lento y las manifestaciones clínicas más frecuentes son obstrucción nasal seguida de epíxtasis. También cefaleas, anosmia y cambios en la visión

Substrate stacking interactions in aryl-alcohol oxidase

This is the peer reviewed version of the following article: [Ferreira, P., Hernández-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martínez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html The version posted may not be updated or replaced with the final published version (the Version of Record).Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2O2 for lignin degradation at the expense of benzylic and other π system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping–pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping–pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state Kd and steady-state Km increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in AAO.This work was supported by the BIO2013-42978-P (to MM), BIO2011-26694 (to ATM), “Juan de la Cierva” (to FL) and CTQ2010-18123 (to VG) Grants of the Spanish Ministry of Economy and Competitiveness (MINECO) and by the INDOX (KBBE-2013-7-613549, to ATM) and PELE (ERC-2009-Adg 25027, to VG) European projects.Peer ReviewedPostprint (author's final draft

Evaluación del rendimiento de simuladores de eventos discretos

El presente trabajo estudia y propone una métrica novedosa para la evaluación del rendimiento de simuladores de eventos discretos. En particular, nos centraremos en la evaluación de simuladores que implementan el formalismo DEVS, y nos basaremos en el enfoque hasta ahora propuesto a través del benchmark DEVStone

A hydrogen bond network in the active site of Anabaena ferredoxin-NADP + reductase modulates its catalytic efficiency

Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP+) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP+ in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event. © 2013 Elsevier B.V.Peer Reviewe

Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals

Protein Motifs Involved in Coenzyme Interaction and Enzymatic Efficiency in Anabaena Ferredoxin-NADP(+) Reductase

Ferredoxin-NADP(+) reductases (FNRs) must determine the coenzyme specificity and allow the transient encounter between N5 of its flavin cofactor and C4 of the coenzyme nicotinamide for efficient hydride transfer. Combined site-directed replacements in different putative determinants of the FNR coenzyme specificity were simultaneously produced. The resulting variants were structurally and functionally analyzed for their binding and hydride transfer abilities to the FNR physiological coenzyme NADP(+)/H, as well as to NAD(+)/H. The previously studied Y303S mutation is the only one that significantly enhances specificity for NAD(+). Combination of mutations from the pyrophosphate or 2'-phosphate regions, even including Y303S, does not improve activity with NAD(+), despite structures of these FNRs show how particular coenzyme-binding regions resembled motifs found in NAD(+)/H-dependent enzymes of the FNR family. Therefore, the "rational approach" did not succeed well, and coenzyme specificity redesign in the FNR family will be more complex than that anticipated in other NADP(+)/NAD(+) families.</p